Use a sterile toothpick or pipette tip to pick an individual colony, and dip into each amplification reaction. To create a stock of each individual colony, either dip the same toothpick or pipette tip into 3 ml growth media containing ampicillin, or use a separate agarose-ampicillin plate to prepare a streak or patch of the colony material.

2a. Transfer the reactions to a thermocycler and perform PCR with the following conditions:

STEP

TEMP

TIME

Initial Denaturation

94°C

2 minutes

30 Cycles

94°C

15 seconds

53–57°C*

15 seconds

68°C

60 seconds/kb

Final Extension

68°C

5 minutes

Hold

4–10°C

∞

*For OneTaq, use 53°C; for LongAmp, use 57°C

Load 5 μl of each completed PCR onto an agarose gel alongside an appropriate DNA ladder [e.g., Quick-Load Purple 2-Log DNA Ladder (NEB #N0550)]. For reference, the amplicon length in the absence of an insertion would be 309 bp in length. The amplicon length with the positive control insert would be 1312 bp.

Screening Protocol 2: Sequence Analysis

The Cloning Analysis Forward and Reverse Primers can also be used for sequencing inserts. This can be performed with purified plasmids from overnight cultures from each colony, or with amplicons from the above colony screening PCRs. The primers anneal 155 bp upsteam and 154 bp downsteam (measured from the 5´ end of each primer to cloning insertion site), ensuring complete reads of the insert.